Significance and molecular targets of protein kinase A during cAMP-mediated protection of cold stored liver grafts

The use of marginal donor livers is followed by a higher frequency of prima ry dys- or nonfunction after transplantation. The present study was designe d to test the hypothesis that stimulation of the cAMP second-messenger sign al pathway might protect the liver from ischemic injury, laying emphasis on the role of protein kinase A-mediated signal transduction. Rat livers were harvested after 45 min of cardiac arrest and preserved in H TK solution for 24 h. Hepatic integrity was assessed thereafter using a blo od-free reperfusion model. Supplementation of the preservation solution with dibutyryl-cAMP (db-cAMP) promoted phosphorylation of BAD at Ser 112 and concomitantly mitigated mito chondrial release of cytochrome c into the cytosol. Apoptotic cell transfor mation was evident in reperfused livers by positive TUNEL-staining of sinus oidal lining cells and the detection of cleaved poly(ADP-ribose) polymerase (PAR-P) in tissue homogenates, by western analysis. Treatment with db-cAMP was effective in minimizing both TUNEL staining and PARP cleavage and sign ificantly reduced postischemic enzyme leakage of alanine aminotransferase t o one half, while hepatic bile production was enhanced by approximately 60% when compared to untreated livers. This functional improvement was accompa nied by a net amelioration of portal vascular conductivity. Inhibition of A kinase-anchoring protein with HT31 completely reversed any of the observed effects obtained by db-cAMP. We conclude that enhancement of cellular cAMP signal maintains hepatic inte grity during and after ischemic preservation which may be attributed to pro tein kinase A dependent phosphorylation of BAD in line with subsequent inhi bition of mitochondria-initiated apoptosis of sinusoidal lining cells.